This Small Business Innovation Research Phase I project will establish feasibility of a novel method for detecting and diagnosing polymicrobial infections in biomedical laboratory animals. Polymicrobial infections in laboratory animals are of significant concern to the research community as they have a demonstrated detrimental effect on experimental outcome. It is therefore paramount to efficiently and accurately identify these microbial pathogens in laboratory animal populations to assure pathogen-free biological resources. While methods for infectious disease detection exist today, their wide- spread use is severely hindered due to their complexity, lack of cost effectiveness, turnaround time, and difficulty in multiplexing. The proposed innovation in this project would alter this reality by streamlining and standardizing the current method into a simple and rapid (<1h) 'one-assay' diagnostic for numerous microbial pathogens to be assayed simultaneously. Our innovative, low-cost approach would reduce the complexity of the diagnostic process and thus be suitable for routine use at animal husbandry sites by less technical personnel. Our process utilizes a new synthetic nucleic acid analogue, PNA, which has demonstrated superior affinity and sequence specificity than both natural nucleic acids and other, more common PNA probe systems. Additionally, PNA probes have the ability to selectively bind to both single and double stranded nucleic acids thus removing the need not only amplify the unknown target, but also removing the need to conduct complicated reverse transcriptase processing. Our platform would likewise be applicable to a wide range of biomedical laboratory animals including aquatic, nonhuman primates, rodent species, and others; and compatible to a wide range of clinical specimens. In this Phase I, we aim to establish the feasibility of a new approach to polymicrobial detection; one that is highly sensitive and multiplexable but equally as important, user-friendly, through standardizing and simplifying the diagnosis process. The specific aims in Phase I, is to establish the feasibility of this new approach by correctly identifying two different Helicobacter species from a solution enriched with a background genomic material. We will further demonstrate this process down to the low concentration of 105 targets/ml. The ultimate goal of this project is to develop a simple, and yet more accurate assay, capable of quickly identifying numerous potential microbial pathogens, in a cost-effective manner and thus suitable for routine use on-site. There are no other methods available that offer the range of capabilities, the simplicity of use and the compatibility to multiple specimens as the assay proposed here.